Development of new genetic tests: atypical cystic fibrosis and HCV genotyping

Recent revolutionary progress in human genomics is reshaping the approach to therapy and diagnosis. Nucleic acid–based testing is the major diagnostic tool not only in the setting of inherited genetic disease but in a wide variety of neoplastic and infectious processes. The “Reverse Line Blot” (RLB) is a robust method for the molecular characterization of several disorders. It is a simple technique that can be used in diagnostic routine also by small laboratories, because the request instruments are few and not expensive. The aim of our project is to exploit the potential of this method for assessment of two different disorders: a rare hereditary disease (atypical cystic fibrosis) and a hepatic pathology caused by the Hepatitis C Virus (HCV). In the first part of the study we explore the potentiality of RLB in detecting known genetic mutations in the human genome and our interest was focused on atypical CF. This pathology is characterized by cystic fibrosis (CF) mild symptoms, involvement of few organs, and only one or none mutations in the CFTR gene. This phenotypic heterogeneity could be explained by modifier genes of the CFTR gene, such as the genes coding for the Epithelial Sodium Channel (ENaC). During this project, we developed a new RLB molecular assay for detection of nine already described variations in the ENaC genes, involved in CF-like disorders. The performance of the method were evaluated analyzing 208 patient samples, with respiratory or pancreatic manifestations, and 169 true healthy individuals as control group. Among the atypical CF patients, with one or none CFTR mutations and showing respiratory symptoms, we found seven ENaC different variants: the mutations p.S82C-SCNN1B, p.P267L-SCNN1B, p.G294S-SCNN1B, p.E539K-SCNN1B, p.1670-2A>G-SCNN1B, and the polymorphisms p.W493R-SCNN1A and p.R181W-SCNN1A. In particular, p.P267L-SCNN1B, p.G294S-SCNN1B, p.E539K-SCNN1B, p.1670-2A>G-SCNN1B showed a frequency significantly higher in patients rather in controls (P